SNX-mediated biogenesis of a plant-unique vesicle derived from the multivesicular body.

Retrograde transport is central to endomembrane homeostasis, yet the identity and origin of plant retrograde carriers remain unresolved. Prevailing models propose that plant vacuolar sorting receptors (VSRs) recycle either from multivesicular bodies (MVBs) to the trans-Golgi network (TGN) or from the TGN to the Golgi apparatus and/or endoplasmic reticulum (ER). However, the ultrastructural features of plant retrograde transport carriers remain largely unresolved. Here, we show that plant retrograde transport is likely mediated by a previously unrecognized class of MVB-derived spherical vesicles. Using correlative light and electron microscopy and three-dimensional electron tomography, we identify a distinct population of ~30-50 nm spherical vesicles adjacent to MVBs, including nascent vesicles budding from the MVB limiting membrane in Arabidopsis root cells. Immunogold labeling shows that these vesicles are enriched in retromer components and VSRs, suggesting that they possibly function as retrograde transport carriers. To investigate their biogenesis, we perform cryo-electron microscopy and liposome tubulation assays, showing that Arabidopsis SNX1 generates shorter membrane tubules than its mammalian counterpart, consistent with reduced membrane affinity linked to differences in the amphipathic helix. Notably, the SNX1-SNX2 heterodimer produces heterogeneous structures, including spherical vesicles, recapitulating in vivo observations. Lastly, knockdown of SNX1 or SNX2 results in vacuolar mislocalization and increased degradation of GFP-VSR2, and defects in SNX1 and VPS29 inhibit formation of spherical vesicles adjacent to MVBs, resulting in embryonic lethality before the globular stage. Together, these findings establish MVB-derived spherical vesicles as plant retrograde carriers and reveal a distinct SNX-mediated mechanism underlying their formation.